Immunomodulatory methods and systems for treatment and/or prevention of atherosclerosis

ABSTRACT

Immunostimulatory methods and systems for treating or preventing atherosclerosis and/or a condition associated thereto in an individual.

CROSS REFERENCE TO RELATED APPLICATIONS

The present disclosure is an application under section 371 ofInternational patent application PCT/US2010/056623 filed Nov. 12, 2010,which claims priority to U.S. provisional application 61/261,331entitled “Immunomodulatory Methods and Systems for Treatment and/orPrevention of Atherosclerosis” filed on Nov. 14, 2009. Each of thesedisclosures are herein incorporated by reference in their entirety.

FIELD

The present disclosure relates to immunomodulatory methods and systemsthat are suitable for treatment or prevention of atherosclerosis and/orconditions associated thereto.

BACKGROUND

Atherosclerosis is currently viewed as a chronic lipid-related andimmune-mediated inflammatory disease of the arterial walls. Many immunecomponents have been identified that participate in atherogenesis.Pre-clinical studies have yielded promising results suggesting thatimmuno-modulatory therapies targeting these components can reduceatherosclerosis.

SUMMARY

Provided herein, are methods and systems for inducing immunomodulatoryresponses in an individual. In several embodiments, the immunomodulatoryresponses induced by the methods and systems of the present disclosureare associated to a therapeutic and/or preventive effect related toatherosclerosis in the individual or a condition associated thereto.

According to a first aspect, a CD8(+) T cell activated by an immunogenicfragment of ApoB100 or an immunogenically active portion thereof isdescribed together with related methods and systems. The activatedCD8(+) T cell is specific for the immunogenic fragment or theimmunogenically active portions thereof.

According to a second aspect, a CD8(+) T cell activated by animmunogenic fragment of ApoB100 or an immunogenically active portionthereof is described for use as a medicament and in particular for usein treatment and/or prevention of atherosclerosis in an individual. Theactivated CD8(+) T cell is specific for the immunogenic fragment or theimmunogenically active portions thereof. The immunogenic fragment orimmunogenically active portion thereof is associated to atherosclerosisreduction in an individual.

According to a third aspect, a method and system to treat and/or preventatherosclerosis in an individual is described. The method comprisesadministering to the individual an effective amount of a CD8(+) Tcellactivated by an immunogenic fragment of ApoB100 or an immunogenicallyactive portion thereof, the immunogenic fragment or immunogenicallyactive portion thereof associated with atherosclerosis reduction, theactivated CD8(+)Tcell being specific for the immunogenic fragment or theimmunogenically active portion thereof. The system comprises at leasttwo of an activated CD8(+) T cell, one or more suitable adjuvants.

According to a fourth aspect, a method and system to provide activatedCD8(+)T cell is described. The method comprises contacting a CD8(+) Tcell with an immunogenic fragment of ApoB100 or an immunogenicallyactive portion thereof for a time and under condition to obtain aCD8(+)Tcell specific for the immunogenic fragment or the immunogenicallyactive portions thereof. In some embodiments, wherein the activatedCD8(+) T cell is suitable to treat and/or prevent atherosclerosis and/ora condition associated thereto. The immunogenic fragment orimmunogenically active portion thereof is associated to atherosclerosisreduction. The system comprises at least two of a CD8(+) T cell, animmunogenic fragment of ApoB100, an immunogenically active portion ofsaid immunogenic fragment, and a T cell enhancer.

According to a fifth aspect, a composition is described comprising atleast one activated CD8(+)T cell herein described together with asuitable vehicle.

According to a sixth aspect, a method and system to identify atherapeutic agent for treatment and/or prevention of atherosclerosis inan individual is described. The method comprises contacting a CD 8(+) Tcell with an immunogenic fragment of ApoB100 or an immunogenicallyactive portion thereof for a time and under condition to provide anactivated CD8(+) T cell, the activated CD8(+)Tcell being specific forthe immunogenic fragment or the immunogenically active portion thereof.The method further comprises administering the activated CD8(+) T cellto an animal model and detecting atherosclerosis reduction in the animalmodel. The system comprises at least two of a CD8(+) T cell, a T cellenhancer, an animal model, reagents to detect T cell activation, andreagents to detect atherosclerosis reduction.

The T cells, compositions, methods and systems herein described can beused in connection with applications wherein reduction of plaque,attenuation of plaque growth and/or a therapeutic or preventive effectfor atherosclerosis in an individual is desired.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description andexamples sections, serve to explain the principles and implementationsof the disclosure.

FIG. 1 shows p210 immunization confers athero-protective effect. (A)Immunization with native p210 resulted in a significant reduction inaortic atherosclerosis when compared to PBS and cBSA/Alum group (n=9-10each group, representative picture from each group shown). (B) P210immunization significantly reduced macrophage infiltration and DCpresence assessed by MOMA-2 (n=9-10 each group) and CD11c (n=7-12 eachgroup) immuno-reactivity, respectively in aortic sinus plaques.

FIG. 2 shows an effect of p210 immunization on DCs. One week afterprimary immunization, (A) CD11c(+) or (B) CD11c(+)CD86(+) cells at theimmunization sites was significantly reduced in p210/cBSA/alum groupwhen compared to cBSA/alum group. N=10 each group. (C) One week afterthird immunization, p210 immunized mice had reduced CD11c(+)CD86(+)cells in lymph nodes compared to cBSA/alum group (n=5 in each group;ANOVA followed by multiple group comparison).

FIG. 3 shows IgM or IgG titer against p210 before and after p210immunization. (A) The p210 IgG titers were low before immunization andremained low in the PBS group at euthanasia but significantly increasedin cBSA/alum and p210/cBSA/alum groups, with the highest titer in thecBSA/alum group. (B) The p210 IgM titers were low before immunizationand significantly increased at euthanasia with no difference among 3groups of mice. N=5 for 6-7 week time-point and n=9 for 25 weektime-point.

FIG. 4 shows activated lymphocyte population after immunization in vivo.(A) CD8(+)CD25(+) T-cell population in the lymph nodes was significantlyhigher in p210/cBSA/alum group when compared to that of PBS or cBSA/alumgroups; (B) CD4(+)CD25(+) T-cells in the lymph nodes did not differamong the three groups. There was a significantly larger population ofsplenic CD8(+)CD25(+)IL-10(+) T-cells in p210/cBSA/alum group among 3groups (C) without difference in splenic CD8(+)CD25(+)IL12(+) T-cellsamong 3 groups (D). Splenic CD4(+)CD25(+)IL-10(+) T-cell populationsignificantly increased in the cBSA/alum group, but was significantlyattenuated by the p210/cBSA/alum immunization (E) and (F) splenicCD4(+)CD25(+)IL12(+) T-cells did not differ among 3 groups. N=9-10 ineach group for (A) and (B); n=5 in each group for (C), (D), (E) and (F).

FIG. 5 shows adoptive transfer of CD8(+) T-cells from p210 immunizeddonors recapitulated the athero-protective effect of p210 immunizationbut not by transfer of B-cells or CD4(+)CD25(+) T-cells. (A) Therecipient mice of CD8(+) T-cells from p210/cBSA/alum immunized donorsdeveloped significantly smaller atherosclerotic lesions compared to therecipient mice of CD8(+) T-cells from other 2 groups (n=9-10 eachgroup). (B) Adoptive transfer of B-cells from p210/cBSA/alum donors didnot reduce atherosclerosis when compared to the recipient mice ofB-cells from PBS or cBSA/alum groups (n=9 each group). Recipient mice ofCD4(+)CD25(+) T-cells (n=9-13 each group) with 2 different doses (C.1×10⁵ cells/mouse or D. 3×10⁵ cells/mouse) did not reproduce theathero-reducing effect of p210 immunization.

FIG. 6 shows increased cytolytic activity of CD8(+) T cells from p210immunized mice against dendritic cells in vitro. CD8(+) T-cells fromp210 immunized mice significantly had a higher cytolytic activityagainst when compared to those from PBS or BSA/alum groups. Experimentswere repeated 4 times with CD8(+) T-cells pooled from 5 mice in eachgroup each time. Duplicate or triplicate was done each time with totalof 11 data-points in each group altogether.

FIG. 7 shows CD8(+) T-cells from p210 immunized mice containing higherlevel of Granzyme B when compared to those from PBS or cBSA/alum group;whereas there is no difference in perforin level

FIG. 8 shows IgG titers against KLH or TNP after p210 immunization. (A)Prior immunization with p210 did not affect the efficacy of subsequentT-cell dependent (KLH, n=3-6 each group) or (B) T-cell independent (TNP,n=4-5 each group) immunization as assessed by the IgG antibody titerswhen compared to mice received PBS or cBSA/alum.

DETAILED DESCRIPTION

T cells and related methods and systems are herein described that aresuitable for treatment and/or prevention of atherosclerosis or acondition associated thereto in an individual

The term “T cells” as used herein indicates T lymphocytes belonging to agroup of white blood cells known as lymphocytes, and participate inhumoral or cell-mediated immunity. T cells can be distinguished fromother lymphocyte types, such as B cells and natural killer cells (NKcells) by the presence of special markers on their cell surface such asT cell receptors (TCR). Additional markers identifying T cell includeCD1a, CD3, CD4, CD8, and additional markers possibly associated to a Tcell state and/or functionality as will be understood by a skilledperson.

The term “treating” or “treatment” as used herein indicates any activitythat is part of a medical care for, or that deals with, a conditionmedically or surgically. The term “preventing” or “prevention” as usedherein indicates any activity, which reduces the burden of mortality ormorbidity from a condition in an individual. This takes place atprimary, secondary and tertiary prevention levels, wherein: a) primaryprevention avoids the development of a disease; b) secondary preventionactivities are aimed at early disease treatment, thereby increasingopportunities for interventions to prevent progression of the diseaseand emergence of symptoms; and c) tertiary prevention reduces thenegative impact of an already established disease by restoring functionand reducing disease-related complications.

The term “atherosclerosis” as used herein indicates a condition in whichan artery wall thickens as the result of a build-up of fatty materialssuch as cholesterol. It is a syndrome typically affecting arterial bloodvessels, in large part due to the accumulation of oxidized lipids,macrophage, activated lymphocytes, and promoted by low-densitylipoproteins (plasma proteins that carry cholesterol and triglycerides).It is commonly referred to as a hardening of the arteries.Atherosclerosis is currently viewed as a chronic lipid-related andimmune-mediated inflammatory disease of the arterial walls. Many immunecomponents have been identified that participate in atherogenesis andpre-clinical studies have yielded promising results suggesting thatimmuno-modulatory therapies targeting these components can reduceatherosclerosis. One of the strategies is to use apoB-100 relatedpeptides as immunogens and several candidate peptides have beenidentified for such use, see references (1) (2).

The term “condition” as used herein indicates as usually the physicalstatus of the body of an individual (as a whole or of one or more of itsparts) that does not conform to a physical status of the individual (asa whole or of one or more of its parts) that is associated with a stateof complete physical, mental and possibly social well-being. Conditionsherein described include but are not limited to disorders and diseaseswherein the term “disorder” indicates a condition of the livingindividual that is associated to a functional abnormality of the body orof any of its parts, and the term “disease” indicates a condition of theliving individual that impairs normal functioning of the body or of anyof its parts and is typically manifested by distinguishing signs andsymptoms. Exemplary conditions include but are not limited to injuries,disabilities, disorders (including mental and physical disorders),syndromes, infections, deviant behaviours of the individual and atypicalvariations of structure and functions of the body of an individual orparts thereof.

The wording “associated to” as used herein with reference to two itemsindicates a relation between the two items such that the occurrence of afirst item is accompanied by the occurrence of the second item, whichincludes but is not limited to a cause-effect relation andsign/symptoms-disease relation.

The term “individuals” as used herein indicates a single biologicalorganism such as higher animals and in particular vertebrates such asmammals and more particularly human beings.

In an embodiment, a CD8(+) Tcell activated by an immunogenic fragment ofApoB100 is described, the activated CD8(+)Tcell specific for theimmunogenic fragment.

The term “CD8(+) T cells” indicates T cells expressing the CD8glycoprotein at their surface, wherein the CD8 (cluster ofdifferentiation 8) glycoprotein is a transmembrane glycoprotein thatserves as a co-receptor for the T cell receptor (TCR). Similarly to theTCR, CD8 binds to a major histocompatibility complex (MHC) molecule, butis specific for the class I MHC protein. Exemplary CD8 T cells comprisecytotoxic memory CD8 T cells, regulatory CD8 T cells, cytotoxic effectorCD8 T-cells and additional cells identifiable by a skilled person. Twoisoforms of the CD8 protein have been identified, alpha and beta, eachencoded by a different gene. In humans, both genes are located onchromosome 2 in position 2p12.

In particular, in some embodiments of the T cell, compositions methodsand systems herein described CD8(+) T cells can be activated using oneor more immunogenic fragments of ApoB100 or an immunogenically activeportion thereof.

The term “activated” and activation as used herein indicate the processby which a T cells interacts with an antigen presenting cell whichpresents a specific antigen for a time and under condition resulting ina T cell having a preassigned immunological role (e.g. cytotoxicity)within the immune system. The term “antigen-presenting cell” (APC)indicates a cell that displays antigen complex with majorhistocompatibility complex (MHC) on its surface. T-cells recognize thiscomplex using their T-cell receptor (TCR). Exemplary APCs comprisedendritic cells (DCs) which are known to play an important role inlinking innate and acquired immunity, see references (3, 4), and bothimmune responses participate in atherogenesis, see references (5), (6).

Detection of T cells and in particular, CD8(+) T cells, can be performedby detection of markers such as CD8, alone or in combination with TCRand additional markers identifiable by a skilled person. Detection ofactivated CD8(+) T cells can be performed by detection of T cellsmarkers and other markers such as CD25, CD44, CD62 and additionalmarkers identifiable by a skilled person using process and techniquessuitable for detecting surface markers.

The terms “detect” or “detection” as used herein indicates thedetermination of the existence, presence or fact of a molecule or cellin a limited portion of space, including but not limited to a sample, areaction mixture, a molecular complex and a substrate. The “detect” or“detection” as used herein can comprise determination of chemical and/orbiological properties of the target, including but not limited toability to interact, and in particular bind, other compounds, ability toactivate another compound and additional properties identifiable by askilled person upon reading of the present disclosure. The detection canbe quantitative or qualitative. A detection is “quantitative” when itrefers, relates to, or involves the measurement of quantity or amount ofthe target or signal (also referred as quantitation), which includes butis not limited to any analysis designed to determine the amounts orproportions of the target or signal. A detection is “qualitative” whenit refers, relates to, or involves identification of a quality or kindof the target or signal in terms of relative abundance to another targetor signal, which is not quantified.

Exemplary techniques suitable for detecting T cell markers comprise useof suitable monoclonal or polyclonal antibodies or antigen-specific HLAor MHC pentamers or hexamers labeled with an appropriate moleculeallowing detection as well as additional methods and techniquesidentifiable by a skilled person. In an exemplary approach T cellmarkers are identified by flow cytometric analysis as described in theExamples section.

Activated CD8(+) T cells according to the present disclosure areactivated with one or more immunogenic fragment of ApoB100 or animmunogenically active portion thereof and are typically specific forthe immunogenic fragment or the immunogenically active portion used forthe activation.

The wording “specific” “specifically” or “specificity” as used hereinwith reference to the immunogenic response refers to the ability of animmunological agent to direct the immunological activity towards anantigen, together with substantially less to no immunological activitytowards other antigen that may be present. As consequence, CD8 (+) Tcells herein are specifically activated towards the immunogenic fragmentor active portion used to activate them and not for other antigens.

Exemplary antigenic properties that can be used to identify CD8 T cellspecific for the immunogenic fragments comprise humoral and/or cellularresponses detectable using methods and techniques such as the onesexemplified in the Examples section as well as other methods andtechniques identifiable by a skilled person. Exemplary methods andsystems for detecting antigenic properties in the sense of the presentdisclosure comprise ELISA and in particular serum ELISA and additionalmethods exemplified in the Examples section. Exemplary techniquessuitable for detecting T cell markers comprise use of suitablemonoclonal or polyclonal antibodies or antigen-specific HLA or MHCpentamers or hexamers labeled with an appropriate molecule allowingdetection as well as additional methods and techniques identifiable by askilled person. In an exemplary approach T cell markers are identifiedby flow cytometric analysis as described in the Examples section.

The term “immunogenic fragment” or “antigenic fragment” as used hereinindicates a portion of a polypeptide of any length capable of generatingan immune response, such as an antigen. An antigen is a moleculerecognized by the immune system. An antigenic fragment of ApoB100 isaccordingly a portion of apoB-100 that presents antigenic properties.

The term “fragment of ApoB100” in the sense of the present disclosurecomprises not only fragments of any length from ApoB100, but alsopeptides produced by genetic recombination or chemically synthesizedcomprising sequences from ApoB100. The term “immunogenic fragments” inthe sense of the present disclosure further comprise also derivative ofany fragment, such as mutated fragments (including fragments withreplaced, added or deleted residues) oxidative derivative and/or peptidetreated with MDA or copper, which maintain a detectable antigenicproperty of the original fragment (e.g. a specific humoral and/orcellular response).

The term “derivative” as used herein with reference to a first peptide(e.g., an immunogenic fragment), indicates a second peptide that isstructurally related to the first peptide and is derivable from thefirst peptide by a modification that introduces a feature that is notpresent in the first peptide while retaining functional properties ofthe first peptide. Accordingly, a derivative peptide of an immunogenicfragment, or of any portion thereof, (e.g. an epitope thereof), usuallydiffers from the original immunogenic fragment or portion thereof bymodification of the amino acidic sequence that might or might not beassociated with an additional function not present in the originalpeptide or portion thereof. A derivative peptide of an immunogenicfragment or of any portion thereof retains however one or more of theimmunogenic activities that are herein described in connection with animmunogenic fragment or portion thereof. The antigenic properties can beverified with methods and systems such as the ones already described forthe immunogenic fragments and additional methods and systemsidentifiable to a skilled person. Typically, a derivative of animmunogenic fragment comprises at least one epitope of the immunogenicfragment

The term “immunogenically active portion” in the sense of the presentdisclosure indicates any part of a reference antigen that can elicitspecific immune response. Exemplary immunogenically active portions areepitopes typically formed by 5 or more residues within an immunogenicfragment. In some embodiments, epitopes within one or more fragments canoverlap.

Immunogenic fragments can be expressed by recombinant technology, suchas a fusion with an affinity or epitope tag, chemical synthesis of anoligopeptide, either free or conjugated to carrier proteins, or anyother methods known in the art to express the ApoB-100 peptides.

Exemplary fragments of ApoB100 are peptides each comprising one of thesequences listed in the Sequence Listing as SEQ ID NO: 1 to SEQ ID NO:302 described in further detail in the Examples section. Methods andsystems suitable to identify an immunogenic fragment in the sense of thepresent are described in WO 02/080954, hereby incorporated by reference.Additional methods are exemplified in the Examples section (see e.g.Example 1).

The term “protein” or “polypeptide” or “peptide” as used hereinindicates an organic polymer composed of two or more amino acid monomersand/or analogs thereof. The term “polypeptide” includes amino acidpolymers of any length including full length proteins or peptides, aswell as analogs and fragments thereof. A peptide of three or more aminoacids is also called an oligopeptide. As used herein the term “aminoacid”, “amino acidic monomer”, or “amino acid residue” refers to any ofthe twenty amino acids including synthetic amino acids with unnaturalside chains and including both D and L optical isomers. The term “aminoacid analog” refers to an amino acid in which one or more individualatoms have been replaced, either with a different atom, isotope, or witha different functional group but is otherwise identical to its naturalamino acid analog.

In an embodiment, the immunogenic fragment of ApoB100 is associated toatherosclerosis reduction. In particular, in some embodiments,immunogenic fragments of ApoB100 are immunogenic fragments producing animmune response associated to atherosclerosis reduction in theindividual or in an animal model. In some of those embodiments, apercentage atherosclerosis reduction is at least 20%, or at least 30% to50% or 40% to 80%. Reference is made to Examples section whereinembodiments of the present disclosure are exemplified with reference toimmunogenic fragment p210 associated with a reduction of atherosclerosisof about 57.6% (see Example 2).

In some embodiments, the immunogenic fragment associated toatherosclerosis reduction comprises at least one of peptide, eachcomprising p1 (SEQ ID NO: 1), p2 (SEQ ID NO: 2), p10 (SEQ ID NO: 10),p11 (SEQ ID NO:11), p25 (SEQ ID NO:25), p30-p34 (SEQ ID NOs:30-34), p40(SEQ ID NO:40), p40 (SEQ ID NO:40), p45 (SEQ ID NO:45), p68 (SEQ IDNO:68), p74 (SEQ ID NO:74), p94 (SEQ ID NO:94), p99 (SEQ ID NO:99), p100(SEQ ID NO:100), p102(SEQ ID NO:102), p103 (SEQ ID NO: 103), p105 (SEQID NO:105), p107 (SEQ ID NO:107), pill (SEQ ID NO:111), p129 (SEQ IDNO:129), p143 (SEQ ID NO:143), p148 (SEQ ID NO:148), p149 (SEQ IDNO:149), p154 (SEQ ID NO:154), p162 (SEQ ID NO:162), p169 (SEQ IDNO:169), p177 (SEQ ID NO:177), p199 (SEQ ID NO:199), p210 (SEQ IDNO:210), p222 (SEQ ID NO:222), p236 (SEQ ID NO:236), p252 (SEQ IDNO:252), or p301 (SEQ ID NO:301).

In an embodiment, the one or more immunogenic fragments associated toatherosclerosis reduction comprises one or more peptides each comprisingp2 (SEQ ID NO:2), p11 (SEQ ID NO:11), p32 (SEQ ID NO:32), p45 (SEQ IDNO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p148 (SEQ IDNO:148), p162 (SEQ ID NO:162), or p210 (SEQ ID NO:210).

In an embodiment, the one or more immunogenic fragments associated toatherosclerosis reduction comprises two peptides each comprising p143(SEQ ID NO: 143), or p210 (SEQ ID NO:210). In an embodiment, the one ormore immunogenic fragments associated to atherosclerosis reductioncomprises three peptides each comprising, one of p11 (SEQ ID NO:11), p25(SEQ ID NO: 25), or p74 (SEQ ID NO:74). In an embodiment, the one ormore immunogenic fragments associated to atherosclerosis reductioncomprises five peptides each comprising one of p99 (SEQ ID NO: 99), p100(SEQ ID NO: 100), p102 (SEQ ID NO: 102), p103 (SEQ ID NO: 103), and p105(SEQ ID NO: 105).

In an embodiment, the one or more immunogenic fragments associated toatherosclerosis reduction comprises one or more peptides each comprisingp2 (SEQ ID NO: 2), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQID NO: 102), or p210 (SEQ ID NO:210).

In an embodiment, the one or more immunogenic fragments associated toatherosclerosis reduction comprise a peptide comprising amino acids16-35 of human apoB-100 (p2; SEQ ID NO:2).

In an embodiment the one or more immunogenic fragments associated toatherosclerosis reduction comprise a peptide comprising amino acids661-680 of human apoB-100 (p45; SEQ ID NO:45).

In an embodiment, the one or more immunogenic fragments associated toatherosclerosis reduction comprise a peptide comprising amino acids3136-3155 of human apoB-100 (P210; SEQ ID NO: 210).

Exemplary data showing association of the above peptides toatherosclerosis reduction are shown in International application WO02/080954, herein incorporated by reference in its entirety (see inparticular Table 1, Table 2, Table A and Table B). In particular forsome of those peptides or combination thereof a percentage reduction of64.6% (p143 and p210), 59.6% (p11, p25 and p74), 56.8% (p129, p148, andp167), p67.7 (p2), 57.9% (p210), 55.2% (p301), 47.4% (p45), 31% (p1) hasbeen detected (see W0/02080954 incorporated herein by reference in itsentirety, and in particular Table B).

In some embodiments, activated CD8(+)T cell suitable herein described,can be activated by contacting a CD8(+) T cell with one or moreimmunogenic fragment of ApoB100, or an immunogenically active portionthereof, for a time and under condition to obtain a CD8(+)Tcell specificfor the immunogenic fragment.

In an embodiment, the contacting can be performed in vivo byadministering an effective amount of immunogenic fragment or of animmunogenically active portion thereof to the individual. In someembodiments, the effective amount can be determined in animal models. Insome embodiment, following the administering, activated T cell areisolated from the individual (e.g. from blood or other fluid samples)according to methods identifiable by a skilled person.

Effective amounts of an immunogenic fragment to activate T cells in vivowill depend on the individual wherein the activation is performed andwill be identifiable by a skilled person. For example in an embodimentthe T cell activation can be performed with an effective amount of fromabout 1 to about 100 μg immunogenic fragment or immunogenically activeportion thereof. In an embodiment, T cell activation can be performedwith an effective amount of from about 1 to about 100 mg immunogenicfragment or immunogenically active portion thereof. Additional effectiveamounts are identifiable by a skilled person in view of the individualwhere activation is performed and the desired activation.

In an embodiment, the contacting can be performed in vitro usingsuitable techniques such as pulsing the T cells with an appropriateamount of immunogenic fragment or active portion thereof (see e.g. (41)and (42) herein incorporated by reference in their entirety).

Effective amounts of an immunogenic fragment to activate T cells invitro are identifiable by a skilled person. For example in an embodimentthe T cell activation can be performed with an effective amount of fromabout 1 to about 100 μg immunogenic fragment or immunogenically activeportion thereof.

In an embodiment, the contacting can be performed by contacting theCD8(+) T cell together with an enhancer of CD8(+) T cell activation

The terms “enhancer” and “enhance” as it pertains to a molecule inconnection with CD8 T cell refers to the ability of a molecule to modifythe immune response by promoting the activation of cells of the immunesystem. The choice of appropriate enhancer can allow control ofactivation of the immune response. Exemplary enhancers include cytokinessuch as IL-2. The term “cytokine” as used herein refers cell signalingmolecules that act as has immunomodulating agents, and comprise proteinssuch as interleukins and interferons as would be identifiable to askilled person. Selection of a suitable cytokine can result underappropriate conditions in the preferential induction of a humoral orcellular immune response.

In an embodiment, the enhancer can be Interleukin 2 (IL2), Interleukin15 (IL-15), TGF-beta (TGF-β), IL2-antiIL-2 antibody complex and/oradditional enhancer identifiable by a skilled person upon reading of thepresent disclosure. Reference is made to the references Mitchell et al2010 (38), Perret et al 2008 (39) and Kamimura et al 2007 (40), eachincorporated by reference in its entirety, which describe exemplary useof enhancer in connection with T cell activation.

In particular in some embodiments, the enhancing is performed byreducing CD86 expression and/or IL12 secretion by dendritic cells in theindividual.

In several embodiments treatment or prevention of atherosclerosis and/ora condition associated thereto in an individual, can be performed byincreasing in the individual activated CD8(+) T cell specific for animmunogenic fragment of ApoB100 or an immunogenically active portionthereof.

In particular, in several embodiments, the increase of p210 specificCD8(+) or other activated CD8(+) T cell herein described is associatedwith a decrease of dendritic cells in the atherosclerotic plaques. Moreparticularly this increase of specific CD8(+) T cell can be performed byadministering to the individual an effective amount of peptide p210,comprising sequence KTTKQ SFDLS VKAQY KKNKH (SEQ ID NO: 210) or anotherimmunogenic fragment herein described associated with atherosclerosisreduction in an individual.

In an embodiment, the increasing can be performed by administering tothe individual an effective amount of an activated CD8(+) T cell.

In an embodiment the effective amount is expected to be comprisedbetween about 500,000 to 2,000,000 cells. In embodiment the effectiveamount is expected to be comprised between about 750,000 to about1,500,000 cells. In an embodiment, the effective amount is expected tobe about 1,000,000 cells.

In particular, in an embodiment administration of about 1,000,000 cellsis expected to result in both treatment and prevention ofatherosclerosis. Administration is expected to be performed inaccordance with dosages and schedule which will be identified based onthe condition of the individual to be treated and the desired effect.For example in administration directed to prevention, administering aneffective amount of activated CD8(+) T cell can performed by performingeither a single administration, or a plurality of administrations (e.g.3 administrations or more, in particular up to 6 administrations) ofactivated CD8(+) T cell herein described in intervals to obtain adesired immunization based on the condition of the individual. Inparticular, a plurality of administrations can be performed whenever aprolonged effect is desired.

Administration of CD8(+) T cell herein described can be performedaccording to methods to immunize an individual identifiable to a skilledperson. In an embodiment, the administering can be performed byparenteral administration. Parenteral administration is a systemic routeof administration where the substance is given by route other than thedigestive tract and includes but is not limited to intravenousadministration, intra-arterial administration, intramuscularadministration, subcutaneous administration, intradermal,administration, intraperitoneal administration, and intravesicalinfusion. In particular, in an embodiment the administering can beperformed by intravenous administration.

In an embodiment, administration can be performed by administeringactivated CD8(+) T cell one time, typically via intravenous route, onetime or multiple times, depending on the desired duration of theimmunization effect.

In an embodiment, administering the CD8(+) T cells is expected to beparticularly effective if performed in combination with one or moreimmunogenic fragments of ApoB100 or an immunogenically active portionthereof alone or together with a CD8(+) T cell enhancer such as IL 2. Inparticular, in some embodiments, the administering can be also performedby administering any combination of the activated CD8(+) T cells hereindescried and any of the immunogenic fragments of ApoB100 hereindescribed possibly in combination with enhancers herein described.

In an embodiment, increasing an activated CD8(+) T cell in theindividual, the activated CD8(+)Tcell specific for an immunogenicfragment of ApoB100 or an immunogenically active portion thereof isexpected to result in reducing plaque, dendritic cell content and/ormacrophage content. According to a possible mechanism provided forguidance purpose only and not intended to be limited, a danger signal(such as oxidized LDL, heat shock protein) in the arterial wall canactivate DCs or another APC, which induce effector T-cells withsubsequent activation of immune-mediated inflammatory cascade andmediators leading to atherosclerotic plaque growth or destabilizationsee references (7), (8). Reduction of such DCs recruitment into arterialwalls can be associated with decreased atherosclerosis.

Also in some embodiments the enhancing is performed by modulatingdendritic cell phenotype in the individual to generate tolerogenicdendritic cells. Additionally in several embodiments, an individualpresenting atherosclerosis and/or a condition associated thereto can betreated by enhancing a T regulatory cell population in the individual.

In an embodiment, CD8(+) T cell can be used in a method to identify atherapeutic agent for treatment and/or prevention of atherosclerosis orof a condition associated thereto. The method comprises contacting aCD8(+) T cell with an immunogenic fragment of ApoB100 or animmunogenically active portion thereof for a time and under condition toprovide an activated CD8(+) T cell, the activated CD8(+)Tcell beingspecific for the immunogenic fragment or the immunogenically activeportion thereof. The method further comprises administering theactivated CD8(+) T cell to an animal model and detecting atherosclerosisreduction in the animal model.

The term “animal model” as used herein indicates a living, non-humananimal used during the research and investigation of human disease, forthe purpose of better understanding the disease without the added riskof causing harm to an actual human being during the process. The animalchosen will usually meet a determined taxonomic equivalency to humans,so as to react to disease or its treatment in a way that resembles humanphysiology as needed. Many drugs, treatments and cures for humandiseases have been developed with the use of animal models. Animalmodels representing specific taxonomic groups in the research and studyof developmental processes are also referred to as model organisms.

In an embodiment, detecting atherosclerosis reduction in the animalmodel can be performed by immunohistochemistry and histomorphometrytechniques such as the ones illustrated in the Examples section.Detecting atherosclerosis reduction in the human can be performed byimaging techniques such as ultrasound determination of carotid intimathickness, MRI scan of carotid atherosclerosis, CT angiogram of coronaryor peripheral arterial circulation.

Immunogenic fragments and/or immunogenically active portion thereof andCD8(+) T cell activated thereby which are associated to a reduction inatherosclerosis following administration in the animal model can beselected as therapeutic agent for treatment or prevention ofatherosclerosis. In particular in some embodiments, immunogenicfragments or an immunogenic portion thereof or a CD8(+) T cell activatedthereby associated to a reduction of at least about 20% atherosclerosiscan be selected as a therapeutic agent. In an embodiment, the percentagereduction can of at about 30% to 50%, or of about 40% to 80%.

As disclosed herein, the CD8(+) Tcell, immunogenic fragments orimmunogenically active portion thereof, and enhancers herein describedcan be provided as a part of systems to treat and/or preventatherosclerosis or of a condition associated thereto or as part of asystem to identify a therapeutic agent in treatment and/or prevention ofatherosclerosis or of a condition associated thereto.

In some embodiments, the system comprises two or more of one or moreagents suitable to increase CD8(+) T cell activated in the individualand/or one or more agents suitable to detect the increased CD8(+) T cellin the individual.

In some embodiments, the system comprises two or more of one or moreagents suitable to increase the activated CD8(+) T cell specific for theimmunogenic fragment of ApoB100 in the individual and one or more agentssuitable to detect the reduced placque, dendritic cell content and/ormacrophage content in the individual.

The systems can be provided in the form of kits of parts. In a kit ofparts, the CD8(+) Tcell herein described and other reagents to performthe method herein described can be comprised in the kit independently.The CD8(+) Tcell herein described can be included in one or morecompositions, and each CD8(+) Tcell herein described can be in acomposition together with a suitable vehicle.

Additional components can include, any of the immunogenic fragmentsherein described or immunogenically active portion thereof alone or incombination, enhancers (e.g. IL2, IL15, IL2-antiIL2 antibody complex),adjuvant, (e.g. aluminum salts and in general aluminum adjuvants),reagents able to detect CD8(+) Tcell herein described, reagents able todetect reduced plaque such as labeled molecules and in particular,labeled antibodies, labels, microfluidic chip, reference standards, andadditional components identifiable by a skilled person upon reading ofthe present disclosure.

The terms “label” and “labeled molecule” as used herein as a componentof a complex or molecule referring to a molecule capable of detection,including but not limited to radioactive isotopes, fluorophores,chemiluminescent dyes, chromophores, enzymes, enzymes substrates, enzymecofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metalsols, ligands (such as biotin, avidin, streptavidin or haptens) and thelike. The term “fluorophore” refers to a substance or a portion thereofwhich is capable of exhibiting fluorescence in a detectable image. As aconsequence, the wording “labeling signal” as used herein indicates thesignal emitted from the label that allows detection of the label,including but not limited to radioactivity, fluorescence,chemiluminescence, production of a compound in outcome of an enzymaticreaction and the like.

In an embodiment, the system is directed to increase CD8(+) T cell in anindividual (e.g. for producing the CD8(+) T cell) and the systemcomprises two or more of one or more agents suitable to increase CD8(+)T cell activated in the individual and/or one or more agents suitable todetect the increased CD8(+) T cell in the individual.

In an embodiment, the system is directed to reduce plaque, dendriticcell content and/or macrophage content (e.g. in an individual fortherapeutic purposes or in an animal model for the purpose of biologicalresearch), and the system comprises two or more of one or more agentssuitable to increase the activated CD8(+) T cell specific for theimmunogenic fragment of ApoB100 or an immunogenically active portionsthereof in the individual and one or more agents suitable to detect thereduced placque, dendritic cell content and/or macrophage content in theindividual.

In some embodiments, detection of a CD8(+) Tcell herein described can becarried either via fluorescent based readouts, in which the labeledantibody is labeled with fluorophore, which includes, but notexhaustively, small molecular dyes, protein chromophores, quantum dots,and gold nanoparticles. Additional techniques are identifiable by askilled person upon reading of the present disclosure and will not befurther discussed in detail.

In particular, the components of the kit can be provided, with suitableinstructions and other necessary reagents, in order to perform themethods here described. The kit will normally contain the compositionsin separate containers. Instructions, for example written or audioinstructions, on paper or electronic support such as tapes or CD-ROMs,for carrying out the assay, will usually be included in the kit. The kitcan also contain, depending on the particular method used, otherpackaged reagents and materials (e.g. wash buffers and the like).

In some embodiments, the CD8(+) Tcell herein described can be includedin compositions together with a suitable vehicle.

The term “vehicle” as used herein indicates any of various media actingusually as solvents, carriers, binders or diluents for T cell comprisedin the composition as an active ingredient.

In some embodiments, where the composition is to be administered to anindividual the composition can be a pharmaceutical anti-inflammatorycomposition, and comprises T cell and a pharmaceutically acceptablevehicle.

In particular, in some embodiments, disclosed are pharmaceuticalcompositions which contain at least one CD8(+) Tcell herein described asherein described, in combination with one or more compatible andpharmaceutically acceptable vehicles, and in particular withpharmaceutically acceptable diluents or excipients. In thosepharmaceutical compositions the CD8(+) Tcell herein described can beadministered as an active ingredient for treatment or prevention of acondition in an individual.

The term “excipient” as used herein indicates an inactive substance usedas a carrier for the active ingredients of a medication. Suitableexcipients for the pharmaceutical compositions herein disclosed includeany substance that enhances the ability of the body of an individual toabsorb a CD8(+) Tcell herein described. Suitable excipients also includeany substance that can be used to bulk up formulations with CD8(+) Tcellherein described to allow for convenient and accurate dosage. Inaddition to their use in the single-dosage quantity, excipients can beused in the manufacturing process to aid in the handling of CD8(+) Tcellherein described. Depending on the route of administration, and form ofmedication, different excipients can be used. Exemplary excipientsinclude but are not limited to antiadherents, binders, coatingsdisintegrants, fillers, flavors (such as sweeteners) and colors,glidants, lubricants, preservatives, sorbents.

The term “diluent” as used herein indicates a diluting agent which isissued to dilute or carry an active ingredient of a composition.Suitable diluent include any substance that can decrease the viscosityof a medicinal preparation.

In an embodiment, compositions herein described can further include anadjuvant. The term “adjuvant” as used herein indicates an agent that canstimulate the immune system and increase the response to a vaccine,without having any specific antigenic effect in itself. The word“adjuvant” comes from the Latin word adjuvare, meaning to help or aid.Typically, an immunologic adjuvant is defined as any substance that actsto accelerate, prolong, or enhance antigen-specific immune responseswhen used in combination with specific vaccine antigens.

Further advantages and characteristics of the present disclosure willbecome more apparent hereinafter from the following detailed disclosureby way of illustration only with reference to an experimental section.

EXAMPLES

The methods and systems herein described are further illustrated in thefollowing examples, which are provided by way of illustration and arenot intended to be limiting.

In particular, the following examples illustrate exemplary CD8(+) T celland related process of activation and use to treat or preventatherosclerosis. More particularly, activation of CD8 (+) T cellperformed with the immunogenic fragment p210 is shown, as well as theuse of the CD8 (+) T cell so obtained to treat and preventatherosclerosis in mice models.

A person skilled in the art will appreciate the applicability and thenecessary modifications to adapt the features described in detail in thepresent section, to additional immunogenic fragments, administeredsubcutaneously or using other routes of administration in vivo or invitro according to embodiments of the present disclosure.

Unless otherwise indicated the following material and methods werefollowed in the Examples reported below.

Selection of Peptides and their Preparation for Immunization

The establishment and screening of human apoB-100 peptides has beenreported (8). Based on Applicants pilot experiments and prior reportssee references (9), (10) Applicants selected peptide 210 (p210, KTTKQSFDLS VKAQY KKNKH—SEQ ID NO: 210) as a candidate immunogen. Native p210peptide (Euro-Diagnostica AB, Sweden) was conjugated to cationic bovineserum albumin (cBSA) as carrier using a method described previously, seereferences (3), (4) Alum was used as adjuvant and mixed withpeptide/cBSA conjugate with 1:1 ratio in volume. Peptide conjugation andmixing with alum were prepared fresh prior to each immunization.

Immunization Protocols

Male apoE (−/−) mice (Jackson Laboratories) were housed in an animalfacility accredited by the American Association of Accreditation ofLaboratory Animal Care and kept on a 12-hour day/night cycle withunrestricted access to water and food. The Institutional Animal Care andUse Committee of Cedars-Sinai Medical Center approved the experimentalprotocols. In a pilot experiment, p210 immunization using 100 μg doseconferred optimum athero-reduction compared to 25 or 50 μg dose. Hence100 μg dose was used for all subsequent experiments. Mice, maintained onnormal chow diet, received subcutaneous primary immunization in thedorsal area between scapulas at 6-7 weeks of age, followed by a boosterat 9 and 12 weeks of age. One week after last booster, diet was switchedto high cholesterol chow (TD 88137, Harlan-Teklad) and continued untileuthanasia at the age of 25 weeks. Separate groups of mice receiving PBSor cBSA/alum at the same immunization time-points served as control.Some mice were sacrificed at 8 or 13 weeks of age to assess immuneresponse against p210.

Tissue Harvesting and Preparation

At euthanasia the hearts were harvested and embedded in OCT compound(Tissue-Tek) for cryo-section. Whole aortas were cleaned, processed andstained with Oil Red O to assess the extent of atherosclerosis en facewith computer-assisted histomorphometry see references (3), (4).

Immunohistochemistry and Histomorphometry

The sections from aortic sinus were stained with MOMA-2 (Serotec), orCD11c (eBioscience) antibody to identify macrophages or dendritic cellsimmunohistochemically using standard protocol. Oil-Red-O stain forplaque size was done using standard protocol. Computer-assistedmorphometric analysis was performed to assess histomorphometry asdescribed previously see references (3), (4).

Serum ELISA

Flat-bottomed 96-well polystyrene plates (MaxiSorp, Germany) werepre-coated with 100 ul (20 μg/ml) p210, KLH, TNP-KLH (BiosearchTechnologies T-5060) or BSA (2 μg/ml for IgG or 10 μg/ml for IgM)respectively by incubation overnight at 4° C. to assess antibodieslevels using standard protocol. The coating concentration was optimizedin pilot experiments. Goat anti-mouse HRP-IgG (Pierce 31437) or IgM(Southern Biotech) were used as detecting antibodies and the boundantibodies were detected by developing in ABTS (Southern Biotech) assubstrate and optical density values were recorded at 405 nm.

Flow Cytometric Analysis

Flow cytometric analysis was performed using standard protocols withantibodies listed in Table 1 below and a FACScan (Becton Dickinson) or aCyAn ADP analyzer (Beckman Coulter). For intracellular cytokinestaining, Brefeldin A (3 μg/ml) was added to the cultured cells for 2hours before cells subject to staining procedure. Cell membranes werepermeabilized for staining intracellular molecules.

TABLE 1 Antigen Clone Type Supplier CD4 GK1.5 FITC-Rat IgG2b, κ BDPharmingen CD8b.2 53-5.8 FITC-Rat IgG1, κ BD Pharmingen CD25 PC61.5PE-Rat IgG1, λ eBioscience IL-10 JES5-16E3 Percp-Cy5.5-Rat eBioscienceIgG2a, κ IL-12 Clone C17.8 Percp-Cy5.5-Rat eBioscience IgG2b, κ CD11cHL3 FITC-Hamster IgG1, λ BD Pharmingen CD86 GL1 PE-Rat IgG2a, κ BDPharmingen TGF-Beta 1D11 APC-Mouse IgG1 R&D system Granzyme B 16G6Alexa-Fluo 647 Rat eBioscience IgG2b, κ Perforin eBioOMAK-D FITC-RatIgG2a, κ eBioscience

Adoptive Transfer Experiment

Male apoE (−/−) mice on regular chow received subcutaneous immunizationas described in previous paragraph and were sacrificed at 13 weeks ofage as donors. Splenocytes from the same treatment group were pooledbefore cell isolation. Donor CD8(+) T-cells, CD4(+)CD25(+) T-cells orB-cells were isolated using Dynabeads FlowComp (Invitrogen) according tothe manufacturer's protocols. CD4(+) T-cells were negatively selectedfrom the splenocytes followed by positive selection of CD4(+)CD25(+)cells. B cells were negatively isolated whereas CD8(+) T-cells werepositively isolated first and released from beads. The purity of pooledCD8(+) T-cells, CD4(+)CD25(+) T-cells and B-cells was 90%, 80% and 70%,respectively. The isolated CD8(+) T-cells (1×10⁶ cells/mouse),CD4(+)CD25(+) T-cells (1×10⁵ or 3×10⁵ cells/mouse) or B-cells (2×10⁷cells/mouse) were then adoptively transferred to naïve male apoE (−/−)recipient mice at 6-7 weeks of age via tail vein injection. In thepublished literatures of vascular biology, the number of adoptivelytransferred lymphocytes varied greatly. For B-cells transfer, the numberof 2×10⁷ cells/mouse was chosen based on two prior reports, seereferences (11), (12). For CD4(+)CD25(+) T-cells transfer, the number ofcells transferred ranged from 5×10⁴ cells/mouse to 1×10⁶ cells/mouse inthe published literature see references (13), (14), (15). HenceApplicants chose 2 intermediate doses for our experiment. As to CD8(+)T-cells, 1×10⁶ cells was chosen based on a report from the field ofautoimmune disease, see reference (16). Applicants did not adoptivelytransferred CD4(+) T-cells because naïve or antigen-primed CD4(+)T-cells are known to be pro-atherogenic, see references. (17), (18).Recipient mice were fed normal chow until 13 weeks of age when chow wasswitched to high cholesterol diet until euthanasia at 25 weeks of age.Aortas were harvested to assess the extent of atherosclerosis.

KLH or Trinitrophenyl-Lipopolysaccharide (TNP-LPS) Immunization

Applicants also tested if p210 immunization affected the efficacy ofsubsequent immunization with other antigens. KLH was chosen as aprototypical T-cell dependent and TNP as a T-cell independent antigen.Male C57/BL6 mice on regular chow received subcutaneous immunizationwith p210 conjugate or adjuvant control as described in previousparagraphs for apoE (−/−) mice. At 13 and 15 weeks of age mice weresubcutaneously immunized with 100 μg KLH (with alum as adjuvant) atinjection sites away from p210 sites or injected intraperitoneally with100 μg TNP-LPS (Sigma). KLH or TNP immunization was done in separategroups of mice. Blood was collected via retro-orbital puncture ateuthanasia (16 weeks of age).

In Vitro Generation of BM-Derived Dendritic Cells (BMDCs)

The method for generating BMDC with GM-CSF was adapted from previouspublication with modification, see reference (19). Briefly, bone marrowcells from femurs and tibiae of male apoE−/− mice were plated into 10 cmculture plates (Falcon) with 20 ml complete RPMI-1640 containing 10ng/ml GM-CSF (R&D Systems) and 10 ng/ml IL-4 (Invitrogen). Cells werewashed and fed on day 3 and day 5 by removing the old medium followed byreplenishing with fresh culture medium with GM-CSF and IL-4. On day 8,the immature DC appeared as non-adherent cells under the microscope andharvested by vigorous pipetting and subcultured into new culture plateswith 2×10⁵ DCs in 1.5 ml medium.

In Vitro CD8+ T-Cells Isolation and Co-Culture with Dendritic Cells

Donor mice [male apoE (−/−) mice] for CD8(+) T-cells were immunized withPBS, cBSA/Alum, or cBSA/Alum/P210 according to the schedule described inearlier paragraphs and splenocytes were harvested at 13 weeks of age.CD8(+) T-cells were negatively isolated using a CD8 selection Dynabeadskit (Invitrogen) per manufacturer's protocol. The selected CD8(+)T-cells were then co-culture with DCs in a CD8:DC ratio of 3:1. A seriesof pilot studies has been performed to determine the optimal CD8:DCratio for this assay. After co-culture for 4 hours, cells were collectedand processed for flow cytometric determination of CD11c and 7-AAD byLSR II flow cytometer (BD Biosciences) and data was analyzed with SummitV4.3 software. Dendritic cell death without CD8(+) T-cells in theco-culture was used as baseline and percentage of specific lysis ofcells was calculated using a method described previously, see reference(20).

Statistics

Data are presented as mean±Std. Number of animals in each group islisted in text or description of the figures. Data were analyzed byANOVA followed by Newman-Keuls multiple group comparison, or by t-testwhen appropriate. P<0.05 was considered as statistically significant andhorizontal bars in each figure indicated statistically significantdifference between groups.

Example 1 Immunogenic Fragments of ApoB-100

Specific immunogenic epitopes by focusing on the single protein found inLDL, apolipoprotein B-100 (apo B) were characterized. A peptide librarycomprised of 302 peptides, 20 amino acid residues in length, coveringthe complete 4563 amino acid sequence of human apo B was produced. Thepeptides were produced with a 5 amino acid overlap to cover allsequences at break points. Peptides were numbered 1-302 starting at theN-terminal of apo B as indicated in Table 2 below.

TABLE 2 Exemplary immunogenic fragments Apolipoprotein Peptide SequenceB aa SEQ ID NO P1: EEEML ENVSL VCPKD ATRFK aa 1-20 SEQ ID NO: 1 P2:ATRFK HLRKY TYNYE AESSS aa 16-35 SEQ ID NO: 2 P3:AESSS GVPGT ADSRS ATRIN aa 31-50 SEQ ID NO: 3 P4:ATRIN CKVEL EVPQL CSFIL aa 46-65 SEQ ID NO: 4 P5:CSFIL KTSQC TLKEV YGFNP aa 61-80 SEQ ID NO: 5 P6:YGFNP EGKAL LKKTK NSEEF aa 76-95 SEQ ID NO: 6 P7:NSEEF AAAMS RYELK LAIPE aa 91-110 SEQ ID NO: 7 P8:LAIPE GKQVF LYPEK DEPTY aa 106-125 SEQ ID NO: 8 P9:DEPTY ILNIK RGIIS ALLVP aa 121-140 SEQ ID NO: 9 P10:ALLVP PETEE AKQVL FLDTV aa 136-155 SEQ ID NO: 10 P11:FLDTV YGNCS THFTV KTRKG aa 151-170 SEQ ID NO: 11 P12:KTRKG NVATE ISTER DLGQC aa 166-185 SEQ ID NO: 12 P13:DLGQC DRFKP IRTGI SPLAL aa 181-200 SEQ ID NO: 13 P14:SPLAL IKGMT RPLST LISSS aa 196-215 SEQ ID NO: 14 P15:LISSS QSCQY TLDAK RKHVA aa 211-230 SEQ ID NO: 15 P16:RKHVA EAICK EQHLF LPFSY aa 226-245 SEQ ID NO: 16 P17:LPFSY NNKYG MVAQV TQTLK aa 241-260 SEQ ID NO: 17 P18:TQTLK LEDTP KINSR FFGEG aa 256-275 SEQ ID NO: 18 P19:FFGEG TKKMG LAFES TKSTS aa 271-290 SEQ ID NO: 19 P20:TKSTS PPKQA EAVLK TLQEL aa 286-305 SEQ ID NO: 20 P21:TLQEL KKLTI SEQNI QRANL aa 301-320 SEQ ID NO: 21 P22:QRANL FNKLV TELRG LSDEA aa 316-335 SEQ ID NO: 22 P23:LSDEA VTSLL PQLIE VSSPI aa 331-350 SEQ ID NO: 23 P24:VSSPI TLQAL VQCGQ PQCST aa 346-365 SEQ ID NO: 24 P25:PQCST HILQW LKRVH ANPLL aa 361-380 SEQ ID NO: 25 P26:ANPLL IDVVT YLVAL IPEPS aa 376-395 SEQ ID NO: 26 P27:IPEPS AQQLR EIFNM ARDQR aa 391-410 SEQ ID NO: 27 P28:ARDQR SRATL YALSH AVNNY aa 406-425 SEQ ID NO: 28 P29:AVNNY HKTNP TGTQE LLDIA aa 421-440 SEQ ID NO: 29 P30:LLDIA NYLME QIQDD CTGDE aa 436-455 SEQ ID NO: 30 P31:CTGDE DYTYL ILRVI GNMGQ aa 451-470 SEQ ID NO: 31 P32:GNMGQ TMEQL TPELK SSILK aa 466-485 SEQ ID NO: 32 P33:SSILK CVQST KPSLM IQKAA aa 481-500 SEQ ID NO: 33 P34:IQKAA IQALR KMEPK DKDQE aa 496-515 SEQ ID NO: 34 P35:DKDQE VLLQT FLDDA SPGDK aa 511-530 SEQ ID NO: 35 P36:SPGDK RLAAY LMLMR SPSQA aa 526-545 SEQ ID NO: 36 P37:SPSQA DINKI VQILP WEQNE aa 541-560 SEQ ID NO: 37 P38:WEQNE QVKNF VASHI ANILN aa 556-575 SEQ ID NO: 38 P39:ANILN SEELD IQDLK KLVKE aa 571-590 SEQ ID NO: 39 P40:KLVKE ALKES QLPTV MDFRK aa 586-605 SEQ ID NO: 40 P41:MDFRK FSRNY QLYKS VSLPS aa 601-620 SEQ ID NO: 41 P42:VSLPS LDPAS AKIEG NLIFD aa 616-635 SEQ ID NO: 42 P43:NLIFD PNNYL PKESM LKTTL aa 631-650 SEQ ID NO: 43 P44:LKTTL TAFGF ASADL IEIGL aa 646-665 SEQ ID NO: 44 P45:IEIGL EGKGF EPTLE ALFGK aa 661-680 SEQ ID NO: 45 P46:ALFGK QGFFP DSVNK ALYWV aa 676-695 SEQ ID NO: 46 P47:ALYWV NGQVP DGVSK VLVDH aa 691-710 SEQ ID NO: 47 P48:VLVDH FGYTK DDKHE QDMVN aa 706-725 SEQ ID NO: 48 P49:QDMVN GIMLS VEKLI KDLKS aa 721-740 SEQ ID NO: 49 P50:KDLKS KEVPE ARAYL RILGE aa 736-755 SEQ ID NO: 50 P51:RILGE ELGFA SLHDL QLLGK aa 751-770 SEQ ID NO: 51 P52:QLLGK LLLMG ARTLQ GIPQM aa 766-785 SEQ ID NO: 52 P53:GIPQM IGEVI RKGSK NDFFL aa 781-800 SEQ ID NO: 53 P54:NDFFL HYIFM ENAFE LPTGA aa 796-815 SEQ ID NO: 54 P55:LPTGA GLQLQ ISSSG VIAPG aa 811-830 SEQ ID NO: 55 P56:VIAPG AKAGV KLEVA NMQAE aa 826-845 SEQ ID NO: 56 P57:NMQAE LVAKP SVSVE FVTNM aa 841-860 SEQ ID NO: 57 P58:FVTNM GIIIP DFARS GVQMN aa 856-875 SEQ ID NO: 58 P59:GVQMN TNFFH ESGLE AHVAL aa 871-890 SEQ ID NO: 59 P60:AHVAL KAGKL KFIIP SPKRP aa 886-905 SEQ ID NO: 60 P61:SPKRP VKLLS GGNTL HLVST aa 901-920 SEQ ID NO: 61 P62:HLVST TKTEV IPPLI ENRQS aa 916-935 SEQ ID NO: 62 P63:ENRQS WSVCK QVFPG LNYCT aa 931-950 SEQ ID NO: 63 P64:LNYCT SGAYS NASST DSASY aa 946-965 SEQ ID NO: 64 P65:DSASY YPLTG DTRLE LELRP aa 961-980 SEQ ID NO: 65 P66:LELRP TGEIE QYSVS ATYEL aa 976-995 SEQ ID NO: 66 P67:ATYEL QREDR ALVDT LKFVT aa 991-1010 SEQ ID NO: 67 P68:LKFVT QAEGA KQTEA TMTFK aa 1006-1025 SEQ ID NO: 68 P69:TMTFK YNRQS MTLSS EVQIP aa 1021-1040 SEQ ID NO: 69 P70:EVQIP DFDVD LGTIL RVNDE aa 1036-1055 SEQ ID NO: 70 P71:RVNDE STEGK TSYRL TLDIQ aa 1051-1070 SEQ ID NO: 71 P72:TLDIQ NKKIT EVALM GHLSC aa 1066-1085 SEQ ID NO: 72 P73:GHLSC DTKEE RKIKG VISIP aa 1081-1100 SEQ ID NO: 73 P74:VISIP RLQAE ARSEI LAHWS aa 1096-1115 SEQ ID NO: 74 P75:LAHWS PAKLL LQMDS SATAY aa 1111-1130 SEQ ID NO: 75 P76:SATAY GSTVS KRVAW HYDEE aa 1126-1145 SEQ ID NO: 76 P77:HYDEE KIEFE WNTGT NVDTK aa 1141-1160 SEQ ID NO: 77 P78:NVDTK KMTSN FPVDL SDYPK aa 1156-1175 SEQ ID NO: 78 P79:SDYPK SLHMY ANRLL DHRVP aa 1171-1190 SEQ ID NO: 79 P80:DHRVP ETDMT FRHVG SKLIV aa 1186-1205 SEQ ID NO: 80 P81:SKLIV AMSSW LQKAS GSLPY aa 1201-1220 SEQ ID NO: 81 P82:GSLPY TQTLQ DHLNS LKEFN aa 1216-1235 SEQ ID NO: 82 P83:LKEFN LQNMG LPDFH IPENL aa 1231-1250 SEQ ID NO: 83 P84:IPENL FLKSD GRVKY TLNKN aa 1246-1260 SEQ ID NO: 84 P85:TLNKN SLKIE IPLPF GGKSS aa 1261-1280 SEQ ID NO: 85 P86:GGKSS RDLKM LETVR TPALH aa 1276-1295 SEQ ID NO: 86 P87:TPALH FKSVG FHLPS REFQV aa 1291-1310 SEQ ID NO: 87 P88:REFQV PTFTI PKLYQ LQVPL aa 1306-1325 SEQ ID NO: 88 P89:LQVPL LGVLD LSTNV YSNLY aa 1321-1340 SEQ ID NO: 89 P90:YSNLY NWSAS YSGGN TSTDH aa 1336-1355 SEQ ID NO: 90 P91:TSTDH FSLRA RYHMK ADSVV aa 1351-1370 SEQ ID NO: 91 P92:ADSVV DLLSY NVQGS GETTY aa 1366-1385 SEQ ID NO: 92 P93:GETTY DHKNT FTLSC DGSLR aa 1381-1400 SEQ ID NO: 93 P94:DGSLR HKFLD SNIKF SHVEK aa 1396-1415 SEQ ID NO: 94 P95:SHVEK LGNNP VSKGL LIFDA aa 1411-1430 SEQ ID NO: 95 P96:LIFDA SSSWG PQMSA SVHLD aa 1426-1445 SEQ ID NO: 96 P97:SVHLD SKKKQ HLFVK EVKID aa 1441-1460 SEQ ID NO: 97 P98:EVKID GQFRV SSFYA KGTYG aa 1456-1475 SEQ ID NO: 98 P99:KGTYG LSCQR DPNTG RLNGE aa 1471-1490 SEQ ID NO: 99 P100:RLNGE SNLRF NSSYL QGTNQ aa 1486-1505 SEQ ID NO: 100 P101:QGTNQ ITGRY EDGTL SLTST aa 1501-1520 SEQ ID NO: 101 P102:SLTST SDLQS GIIKN TASLK aa 1516-1535 SEQ ID NO: 102 P103:TASLK YENYE LTLKS DTNGK aa 1531-1550 SEQ ID NO: 103 P104:DTNGK YKNFA TSNKM DMTFS aa 1546-1565 SEQ ID NO: 104 P105:DMTFS KQNAL LRSEY QADYE aa 1561-1580 SEQ ID NO: 105 P106:QADYE SLRFF SLLSG SLNSH aa 1576-1595 SEQ ID NO: 106 P107:SLNSH GLELN ADILG TDKIN aa 1591-1610 SEQ ID NO: 107 P108:TDKIN SGAHK ATLRI GQDGI aa 1606-1625 SEQ ID NO: 108 P109:GQDGI STSAT TNLKC SLLVL aa 1621-1640 SEQ ID NO: 109 P110:SLLVL ENELN AELGL SGASM aa 1636-1655 SEQ ID NO: 110 P111:SGASM KLTTN GRFRE HNAKF aa 1651-1670 SEQ ID NO: 111 P112:HNAKF SLDGK AALTE LSLGS aa 1666-1685 SEQ ID NO: 112 P113:LSLGS AYQAM ILGVD SKNIF aa 1681-1700 SEQ ID NO: 113 P114:SKNIF NFKVS QEGLK LSNDM aa 1696-1715 SEQ ID NO: 114 P115:LSNDM MGSYA EMKFD HTNSL aa 1711-1730 SEQ ID NO: 115 P116:HTNSL NIAGL SLDFS SKLDN aa 1726-1745 SEQ ID NO: 116 P117:SKLDN IYSSD KFYKQ TVNLQ aa 1741-1760 SEQ ID NO: 117 P118:TVNLQ LQPYS LVTTL NSDLK aa 1756-1775 SEQ ID NO: 118 P119:NSDLK YNALD LTNNG KLRLE aa 1771-1790 SEQ ID NO: 119 P120:KLRLE PLKLH VAGNL KGAYQ aa 1786-1805 SEQ ID NO: 120 P121:KGAYQ NNEIK HIYAI SSAAL aa 1801-1820 SEQ ID NO: 121 P122:SSAAL SASYK ADTVA KVQGV aa 1816-1835 SEQ ID NO: 122 P123:KVQGV EFSHR LNTDI AGLAS aa 1831-1850 SEQ ID NO: 123 P124:AGLAS AIDMS TNYNS DSLHF aa 1846-1865 SEQ ID NO: 124 P125:DSLHF SNVFR SVMAP FTMTI aa 1861-1880 SEQ ID NO: 125 P126:FTMTI DAHTN GNGKL ALWGE aa 1876-1895 SEQ ID NO: 126 P127:ALWGE HTGQL YSKFL LKAEP aa 1891-1910 SEQ ID NO: 127 P128:LKAEP LAFTF SHDYK GSTSH aa 1906-1925 SEQ ID NO: 128 P129:GSTSH HLVSR KSISA ALEHK aa 1921-1940 SEQ ID NO: 129 P130:ALEHK VSALL TPAEQ TGTWK aa 1936-1955 SEQ ID NO: 130 P131:TGTWK LKTQF NNNEY SQDLD aa 1951-1970 SEQ ID NO: 131 P132:SQDLD AYNTK DKIGV ELTGR aa 1966-1985 SEQ ID NO: 132 P133:ELTGR TLADL TLLDS PIKVP aa 1981-2000 SEQ ID NO: 133 P134:PIKVP LLLSE PINII DALEM aa 1996-2015 SEQ ID NO: 134 P135:DALEM RDAVE KPQEF TIVAF aa 2011-2030 SEQ ID NO: 135 P136:TIVAF VKYDK NQDVH SINLP aa 2026-2045 SEQ ID NO: 136 P137:SINLP FFETL QEYFE RNRQT aa 2041-2060 SEQ ID NO: 137 P138:RNRQT IIVVV ENVQR NLKHI aa 2056-2075 SEQ ID NO: 138 P139:NLKHI NIDQF VRKYR AALGK aa 2071-2090 SEQ ID NO: 139 P140:AALGK LPQQA NDYLN SFNWE aa 2086-2105 SEQ ID NO: 140 P141:SFNWE RQVSH AKEKL TALTK aa 2101-2120 SEQ ID NO: 141 P142:TALTK KYRIT ENDIQ IALDD aa 2116-2135 SEQ ID NO: 142 P143:IALDD AKINF NEKLS QLQTY aa 2131-2150 SEQ ID NO: 143 P144:QLQTY MIQFD QYIKD SYDLH aa 2146-2165 SEQ ID NO: 144 P145:SYDLH DLKIA IANII DEIIE aa 2161-2180 SEQ ID NO: 145 P146:DEIIE KLKSL DEHYH IRVNL aa 2176-2195 SEQ ID NO: 146 P147:IRVNL VKTIH DLHLF IENID aa 2191-2210 SEQ ID NO: 147 P148:IENID FNKSG SSTAS WIQNV aa 2206-2225 SEQ ID NO: 148 P149:WIQNV DTKYQ IRIQI QEKLQ aa 2221-2240 SEQ ID NO: 149 P150:QEKLQ QLKRH IQNID IQHLA aa 2236-2255 SEQ ID NO: 150 P151:IQHLA GKLKQ HIEAI DVRVL aa 2251-2270 SEQ ID NO: 151 P152:DVRVL LDQLG TTISF ERIND aa 2266-2285 SEQ ID NO: 152 P153:ERIND VLEHV KHFVI NLIGD aa 2281-2300 SEQ ID NO: 153 P154:NLIGD FEVAE KINAF RAKVH aa 2296-2315 SEQ ID NO: 154 P155:RAKVH ELIER YEVDQ QIQVL aa 2311-2330 SEQ ID NO: 155 P156:QIQVL MDKLV ELTHQ YKLKE aa 2326-2345 SEQ ID NO: 156 P157:YKLKE TIQKL SNVLQ QVKIK aa 2341-2360 SEQ ID NO: 157 P158:QVKIK DYFEK LVGFI DDAVK aa 2356-2375 SEQ ID NO: 158 P159:DDAVK KLNEL SFKTF IEDVN aa 2371-2390 SEQ ID NO: 159 P160:IEDVN KFLDM LIKKL KSFDY aa 2386-2405 SEQ ID NO: 160 P161:KSFDY HQFVD ETNDK IREVT aa 2401-2420 SEQ ID NO: 161 P162:IREVT QRLNG EIQAL ELPQK aa 2416-2435 SEQ ID NO: 162 P163:ELPQK AEALK LFLEE TKATV aa 2431-2450 SEQ ID NO: 163 P164:TKATV AVYLE SLQDT KITLI aa 2446-2465 SEQ ID NO: 164 P165:KITLI INWLQ EALSS ASLAH aa 2461-2480 SEQ ID NO: 165 P166:ASLAH MKAKF RETLE DTRDR aa 2476-2495 SEQ ID NO: 166 P167:DTRDR MYQMD IQQEL QRYLS aa 2491-2510 SEQ ID NO: 167 P168:QRYLS LVGQV YSTLV TYISD aa 2506-2515 SEQ ID NO: 168 P169:TYISD WWTLA AKNLT DFAEQ aa 2521-2540 SEQ ID NO: 169 P170:DFAEQ YSIQD WAKRM KALVE aa 2536-2555 SEQ ID NO: 170 P171:KALVE QGFTV PEIKT ILGTM aa 2551-2570 SEQ ID NO: 171 P172:ILGTM PAFEV SLQAL QKATF aa 2566-2585 SEQ ID NO: 172 P173:QKATF QTPDF IVPLT DLRIP aa 2581-2600 SEQ ID NO: 173 P174:DLRIP SVQIN FKDLK NIKIP aa 2596-2615 SEQ ID NO: 174 P175:NIKIP SRFST PEFTI LNTFH aa 2611-2630 SEQ ID NO: 175 P176:LNTFH IPSFT IDFVE MKVKI aa 2626-2645 SEQ ID NO: 176 P177:MKVKI IRTID QMQNS ELQWP aa 2641-2660 SEQ ID NO: 177 P178:ELQWP VPDIY LRDLK VEDIP aa 2656-2675 SEQ ID NO: 178 P179:VEDIP LARIT LPDFR LPEIA aa 2671-2690 SEQ ID NO: 179 P180:LPEIA IPEFI IPTLN LNDFQ aa 2686-2705 SEQ ID NO: 180 P181:LNDFQ VPDLH IPEFQ LPHIS aa 2701-2720 SEQ ID NO: 181 P182:LPHIS HTIEV PTFGK LYSIL aa 2716-2735 SEQ ID NO: 182 P183:LYSIL KIQSP LFTLD ANADI aa 2731-2750 SEQ ID NO: 183 P184:ANADI GNGTT SANEA GIAAS aa 2746-2765 SEQ ID NO: 184 P185:GIAAS ITAKG ESKLE VLNFD aa 2761-2780 SEQ ID NO: 185 P186:VLNFD FQANA QLSNP KINPL aa 2776-2795 SEQ ID NO: 186 P187:KINPL ALKES VKFSS KYLRT aa 2791-2810 SEQ ID NO: 187 P188:KYLRT EHGSE MLFFG NAIEG aa 2806-2825 SEQ ID NO: 188 P189:NAIEG KSNTV ASLHT EKNTL aa 2821-2840 SEQ ID NO: 189 P190:EKNTL ELSNG VIVKI NNQLT aa 2836-2855 SEQ ID NO: 190 P191:NNQLT LDSNT KYFHK LNIPK aa 2851-2870 SEQ ID NO: 191 P192:LNIPK LDFSS QADLR NEIKT aa 2866-2885 SEQ ID NO: 192 P193:NEIKT LLKAG HIAWT SSGKG aa 2881-2900 SEQ ID NO: 193 P194:SSGKG SWKWA CPRFS DEGTH aa 2896-2915 SEQ ID NO: 194 P195:DEGTH ESQIS FTIEG PLTSF aa 2911-2930 SEQ ID NO: 195 P196:PLTSF GLSNK INSKH LRVNQ aa 2926-2945 SEQ ID NO: 196 P197:LRVNQ NLVYE SGSLN FSKLE aa 2941-2960 SEQ ID NO: 197 P198:FSKLE IQSQV DSQHV GHSVL aa 2956-2975 SEQ ID NO: 198 P199:GHSVL TAKGM ALFGE GKAEF aa 2971-2990 SEQ ID NO: 199 P200:GKAEF TGRHD AHLNG KVIGT aa 2986-3005 SEQ ID NO: 200 P201:KVIGT LKNSL FFSAQ PFEIT aa 3001-3020 SEQ ID NO: 201 P202:PFEIT ASTNN EGNLK VRFPL aa 3016-3035 SEQ ID NO: 202 P203:VRFPL RLTGK IDFLN NYALF aa 3031-3050 SEQ ID NO: 203 P204:NYALF LSPSA QQASW QVSAR aa 3046-3065 SEQ ID NO: 204 P205:QVSAR FNQYK YNQNF SAGNN aa 3061-3080 SEQ ID NO: 205 P206:SAGNN ENIME AHVGI NGEAN aa 3076-3095 SEQ ID NO: 206 P207:NGEAN LDFLN IPLTI PEMRL aa 3091-3110 SEQ ID NO: 207 P208:PEMRL PYTII TTPPL KDFSL aa 3106-3125 SEQ ID NO: 208 P209:KDFSL WEKTG LKEFL KTTKQ aa 3121-3140 SEQ ID NO: 209 P210:KTTKQ SFDLS VKAQY KKNKH aa 3136-3155 SEQ ID NO: 210 P211:KKNKH RHSIT NPLAV LCEFI aa 3151-3170 SEQ ID NO: 211 P212:LCEFI SQSIK SFDRH FEKNR aa 3166-3185 SEQ ID NO: 212 P213:FEKNR NNALD FVTKS YNETK aa 3181-3200 SEQ ID NO: 213 P214:YNETK IKFDK YKAEK SHDEL aa 3196-3215 SEQ ID NO: 214 P215:SHDEL PRTFQ IPGYT VPVVN aa 3211-3230 SEQ ID NO: 215 P216:VPVVN VEVSP FTIEM SAFGY aa 3226-3245 SEQ ID NO: 216 P217:SAFGY VFPKA VSMPS FSILG aa 3241-3260 SEQ ID NO: 217 P218:FSILG SDVRV PSYTL ILPSL aa 3256-3275 SEQ ID NO: 218 P219:ILPSL ELPVL HVPRN LKLSL aa 3271-3290 SEQ ID NO: 219 P220:LKLSL PHFKE LCTIS HIFIP aa 3286-3305 SEQ ID NO: 220 P221:HIFIP AMGNI TYDFS FKSSV aa 3301-3320 SEQ ID NO: 221 P222:FKSSV ITLNT NAELF NQSDI aa 3316-3335 SEQ ID NO: 222 P223:NQSDI VAHLL SSSSS VIDAL aa 3331-3350 SEQ ID NO: 223 P224:VIDAL QYKLE GTTRL TRKRG aa 3346-3365 SEQ ID NO: 224 P225:TRKRG LKLAT ALSLS NKFVE aa 3361-3380 SEQ ID NO: 225 P226:NKFVE GSHNS TVSLT TKNME aa 3376-3395 SEQ ID NO: 226 P227:TKNME VSVAK TTKAE IPILR aa 3391-3410 SEQ ID NO: 227 P228:IPILR MNFKQ ELNGN TKSKP aa 3406-3425 SEQ ID NO: 228 P229:TKSKP TVSSS MEFKY DFNSS aa 3421-3440 SEQ ID NO: 229 P230:DFNSS MLYST AKGAV DHKLS aa 3436-3455 SEQ ID NO: 230 P231:DHKLS LESLT SYFSI ESSTK aa 3451-3470 SEQ ID NO: 231 P232:ESSTK GDVKG SVLSR EYSGT aa 3466-3485 SEQ ID NO: 232 P233:EYSGT IASEA NTYLN SKSTR aa 3481-3500 SEQ ID NO: 233 P234:SKSTR SSVKL QGTSK IDDIW aa 3496-3515 SEQ ID NO: 234 P235:IDDIW NLEVK ENFAG EATLQ aa 3511-3530 SEQ ID NO: 235 P236:EATLQ RIYSL WEHST KNHLQ aa 3526-3545 SEQ ID NO: 236 P237:KNHLQ LEGLF FTNGE HTSKA aa 3541-3560 SEQ ID NO: 237 P238:HTSKA TLELS PWQMS ALVQV aa 3556-3575 SEQ ID NO: 238 P239:ALVQV HASQP SSFHD FPDLG aa 3571-3590 SEQ ID NO: 239 P240:FPDLG QEVAL NANTK NQKIR aa 3586-3605 SEQ ID NO: 240 P241:NQKIR WKNEV RIHSG SFQSQ aa 3601-3620 SEQ ID NO: 241 P242:SFQSQ VELSN DQEKA HLDIA aa 3616-3635 SEQ ID NO: 242 P243:HLDIA GSLEG HLRFL KNIIL aa 3631-3650 SEQ ID NO: 243 P244:KNIIL PVYDK SLWDF LKLDV aa 3646-3665 SEQ ID NO: 244 P245:LKLDV TTSIG RRQHL RVSTA aa 3661-3680 SEQ ID NO: 245 P246:RVSTA FVYTK NPNGY SFSIP aa 3676-3695 SEQ ID NO: 246 P247:SFSIP VKVLA DKFIT PGLKL aa 3691-3710 SEQ ID NO: 247 P248:PGLKL NDLNS VLVMP TFHVP aa 3706-3725 SEQ ID NO: 248 P249:TFHVP FTDLQ VPSCK LDFRE aa 3721-3740 SEQ ID NO: 249 P250:LDFRE IQIYK KLRTS SFALN aa 3736-3755 SEQ ID NO: 250 P251:SFALN LPTLP EVKFP EVDVL aa 3751-3770 SEQ ID NO: 251 P252:EVDVL TKYSQ PEDSL IPFFE aa 3766-3785 SEQ ID NO: 252 P253:IPFFE ITVPE SQLTV SQFTL aa 3781-3800 SEQ ID NO: 253 P254:SQFTL PKSVS DGIAA LDLNA aa 3796-3815 SEQ ID NO: 254 P255:LDLNA VANKI ADFEL PTIIV aa 3811-3830 SEQ ID NO: 255 P256:PTIIV PEQTI EIPSI KFSVP aa 3826-3845 SEQ ID NO: 256 P257:KFSVP AGIVI PSFQA LTARF aa 3841-3860 SEQ ID NO: 257 P258:LTARF EVDSP VYNAT WSASL aa 3856-3875 SEQ ID NO: 258 P259:WSASL KNKAD YVETV LDSTC aa 3871-3890 SEQ ID NO: 259 P260:LDSTC SSTVQ FLEYE LNVLG aa 3886-3905 SEQ ID NO: 260 P261:LNVLG THKIE DGTLA SKTKG aa 3901-3920 SEQ ID NO: 261 P262:SKTKG TLAHR DFSAE YEEDG aa 3916-3935 SEQ ID NO: 262 P263:YEEDG KFEGL QEWEG KAHLN aa 3931-3950 SEQ ID NO: 263 P264:KAHLN IKSPA FTDLH LRYQK aa 3946-3965 SEQ ID NO: 264 P265:LRYQK DKKGI STSAA SPAVG aa 3961-3980 SEQ ID NO: 265 P266:SPAVG TVGMD MDEDD DFSKW aa 3976-3995 SEQ ID NO: 266 P267:DFSKW NFYYS PQSSP DKKLT aa 3991-4010 SEQ ID NO: 267 P268:DKKLT IFKTE LRVRE SDEET aa 4006-4025 SEQ ID NO: 268 P269:SDEET QIKVN WEEEA ASGLL aa 4021-4040 SEQ ID NO: 269 P270:ASGLL TSLKD NVPKA TGVLY aa 4036-4055 SEQ ID NO: 270 P271:TGVLY DYVNK YHWEH TGLTL aa 4051-4070 SEQ ID NO: 271 P272:TGLTL REVSS KLRRN LQNNA aa 4066-4085 SEQ ID NO: 272 P273:LQNNA EWVYQ GAIRQ IDDID aa 4081-4100 SEQ ID NO: 273 P274:IDDID VRFQK AASGT TGTYQ aa 4096-4115 SEQ ID NO: 274 P275:TGTYQ EWKDK AQNLY QELLT aa 4111-4130 SEQ ID NO: 275 P276:QELLT QEGQA SFQGL KDNVF aa 4126-4145 SEQ ID NO: 276 P277:KDNVF DGLVR VTQKF HMKVK aa 4141-4160 SEQ ID NO: 277 P278:HMKVK HLIDS LIDFL NFPRF aa 4156-4175 SEQ ID NO: 278 P279:NFPRF QFPGK PGIYT REELC aa 4171-4190 SEQ ID NO: 279 P280:REELC TMFIR EVGTV LSQVY aa 4186-4205 SEQ ID NO: 280 P281:LSQVY SKVHN GSEIL FSYFQ aa 4201-4220 SEQ ID NO: 281 P282:FSYFQ DLVIT LPFEL RKHKL aa 4216-4235 SEQ ID NO: 282 P283:RKHKL IDVIS MYREL LKDLS aa 4231-4250 SEQ ID NO: 283 P284:LKDLS KEAQE VFKAI QSLKT aa 4246-4265 SEQ ID NO: 284 P285:QSLKT TEVLR NLQDL LQFIF aa 4261-4280 SEQ ID NO: 285 P286:LQFIF QLIED NIKQL KEMKF aa 4276-4295 SEQ ID NO: 286 P287:KEMKF TYLIN YIQDE INTIF aa 4291-4310 SEQ ID NO: 287 P288:INTIF NDYIP YVFKL LKENL aa 4306-4325 SEQ ID NO: 288 P289:LKENL CLNLH KFNEF IQNEL aa 4321-4340 SEQ ID NO: 289 P290:IQNEL QEASQ ELQQI HQYIM aa 4336-4355 SEQ ID NO: 290 P291:HQYIM ALREE YFDPS IVGWT aa 4351-4370 SEQ ID NO: 291 P292:IVGWT VKYYE LEEKI VSLIK aa 4366-4385 SEQ ID NO: 292 P293:VSLIK NLLVA LKDFH SEYIV aa 4381-4400 SEQ ID NO: 293 P294:SEYIV SASNF TSQLS SQVEQ aa 4396-4415 SEQ ID NO: 294 P295:SQVEQ FLHRN IQEYL SILTD aa 4411-4430 SEQ ID NO: 295 P296:SILTD PDGKG KEKIA ELSAT aa 4426-4445 SEQ ID NO: 296 P297:ELSAT AQEII KSQAI ATKKI aa 4441-4460 SEQ ID NO: 297 P298:TKKII SDYHQ QFRYK LQDFS aa 4457-4476 SEQ ID NO: 298 P299:LQDFS DQLSD YYEKF IAESK aa 4472-4491 SEQ ID NO: 299 P300:IAESK RLIDL SIQNY HTFLI aa 4487-4506 SEQ ID NO: 300 P301:HTFLI YITEL LKKLQ STTVM aa 4502-4521 SEQ ID NO: 301 P302:STTVM NPYMK LAPGE LTIIL aa 4517-4536 SEQ ID NO: 302

The full length sequence of ApoB100 can be found in various publicationssuch as reference (43) (see in particular FIG. 1) herein incorporated byreference in its entirety.

Example 2 Athero-Protective Effects of p210 Immunization

Immunization with p210 reduced aortic atherosclerosis by 57% and 50%compared to PBS and cBSA/Alum group, respectively (FIG. 1A) withoutaffecting circulating cholesterol levels or body weight (Table 3).

TABLE 3 Circulating level of cholesterol and body weight of mice fromPBS, cBSA/alum and p210/cBSA/alum group PBS cBSA/alum P210/cBSA/alum Pvalue (n = 10) (n = 10) (n = 10) (ANOVA) Cholesterol 1503 ± 485 1395 ±420 1135 ± 382 0.17 (mg/dl) Body 37.9 ± 5.4 34.8 ± 5.4 34.3 ± 6.5 0.33weight (gm)

The aortic sinus plaques from p210/cBSA/alum group containedsignificantly reduced macrophage and DC immuno-reactivity assessed byMOMA-2 and CD11c immuno-staining, respectively (FIG. 1B).

Example 3 Characterization of p210-Immunization Elicited ImmuneResponses

Since DCs are the major cell type upstream to both cellular and humoralimmune responses, Applicants determined if these cells were affected bythe immunization strategy. Cells from the subcutaneous immunizationsites were isolated for flow cytometric analysis one week after primaryimmunization. The PBS group could not be included in this analysisbecause mice receiving PBS injection did not develop swelling or cellaccumulation at the injection site.

There were significantly fewer CD11c(+) and CD11c(+)CD86(+) cells inp210/cBSA/alum group compared to cBSA/alum group at the immunizationsite (FIGS. 2A and 2B). When flow cytometry was performed on LN cells 1week after the third immunization, CD11c(+)CD86(+) cells were alsosignificantly reduced compared with cBSA/alum group (FIG. 2C).

Applicants next assessed antibody response to define the humoral immuneresponse against p210. Before immunization all 3 groups of mice had lowlevels of IgG titers against p210. At euthanasia, the IgG titer againstp210 remained low in the PBS group but was significantly increased incBSA/alum group. Immunization with p210/cBSA/alum resulted in increasedp210 IgG titer compared with PBS group but was significantly reducedcompared with cBSA/alum group (FIG. 3A). In contrast to p210 IgGresponse, there was a significant increase in p210 IgM titer in allgroups (FIG. 3B), suggesting an endogenous immune response against p210.

The IL-2Rα (CD25) is a well-defined lymphocyte activation marker.Applicants therefore analyzed the expression of CD25 on CD4(+) or CD8(+)T-cells from superficial cervical and axillary lymph nodes (LN) frommice one week after primary immunization to assess the T-cell immuneresponse. CD8(+)CD25(+) T-cell population in the lymph nodes wassignificantly higher in p210/cBSA/alum group when compared to that ofPBS or cBSA/alum groups (FIG. 4A) whereas CD4(+)CD25(+) T-cells in thelymph nodes (FIG. 4B) did not differ among 3 groups.

There was a significantly larger population of splenicCD8(+)CD25(+)IL-10(+) T-cells in p210/cBSA/alum group when compared toPBS or cBSA/alum groups (FIG. 4C) without difference in splenicCD8(+)CD25(+)IL12(+) T-cells among 3 groups (FIG. 4D). SplenicCD4(+)CD25(+)IL-10(+) T-cell population significantly increased in thecBSA/alum group. However, this increased response was significantlyattenuated by the p210/cBSA/alum immunization (FIG. 4E); whereas splenicCD4(+)CD25(+)IL12(+) T-cells did not differ among the three groups (FIG.4F).

Example 4 Adoptive Transfer of CD8(+) T-Cells from p210 Immunized Miceto Naive Recipients Recapitulates the Athero-Protective Effect of p210Immunization

Donor CD8(+)T-cells from PBS, cBSA/alum or p210/cBSA/alum groups wereadoptively transferred to 6-7 week-old unimmunized recipient apoE (−/−)mice. At euthanasia, the recipient mice injected with CD8(+) T-cellsfrom p210/cBSA/alum group developed significantly less atheroscleroticlesions in aorta compared to the recipient mice injected with CD8(+)T-cells from PBS or cBSA/alum groups (FIG. 5A).

This reduction of aortic lesions was coupled with decreased splenicCD11c(+) DCs (PBS group: 4.3±1.7%; cBSA/alum group: 3.4±0.3%;p210/cBSA/alum group: 1.5±0.3%; n=5 each group, p<0.05 p210/cBSA/alumgroup vs. PBS or cBSA/alum group by ANOVA) with no difference incirculating levels of total cholesterol among 3 groups (PBS group:1083±296 mg/dl; cBSA/alum group: 975±401 mg/dl; p210/cBSA/alum group:1098±379 mg/dl).

Adoptive transfer of B cells isolated from the spleens of p210 immunizeddonor mice did not affect atherosclerosis in recipient mice compared tomice receiving B cells from other donors (FIG. 5B).

To rule out CD4(+)CD25(+) T-cells as possible athero-protectivemediators induced by sub-cutaneous p210 immunization, Applicantsadoptively transferred CD4(+)CD25(+)T-cells at a dose of 1×10⁵cells/mouse into naïve recipient apoE−/− mice. There was no differencein lesion size among the 3 groups of CD4(+)CD25(+)T-cell recipients(FIG. 5C). Transfer of a higher number of CD4(+)CD25(+) T-cells at 3×10⁵cells/mouse did not reduce lesion sizes in all 3 recipient groups (FIG.5D).

Example 5 Increased Cytolytic Activity of CD8(+) T Cells from p210Immunized Mice Against Dendritic Cells In Vitro

Given the observation that p210 immunization reduced DCs in theimmunization sites and atherosclerotic plaques and adoptive transfer ofCD8(+) T-cells from p210 immunized donors rendered a decrease of splenicDCs in the recipients, Applicants hypothesized that DCs could be apotential target of CD8(+) T-cells.

To test this, Applicants co-cultured bone marrow derived DCs with CD8(+)T-cells from various immunized groups. CD8(+) T-cells from p210immunized mice significantly increased the percentage of DC death whencompared to those from PBS or BSA/alum groups (FIG. 6). This increasedcytolytic function of CD8(+) T-cells was associated with increasedgranzyme B expression but not perforin (FIG. 7).

Example 6 Immunization with p210 does not Affect the Adaptive ImmuneResponse to Other T-Cell Dependent or Independent Antigens

Given the observations that p210 immunization decreased CD11c(+) DCs andreduced adaptive IgG response to p210, Applicants next tested if suchmodulation of DCs by p210 immunization would alter the host immuneresponse to other antigens.

Applicants first immunized mice with p210 as described in previoussections followed by two separate subcutaneous KLH immunizations orintra-peritoneal injection of TNP-LPS. Using the KLH- or TNP-IgG titeras a surrogate for the efficacy of individual immunization, Applicantsfound that there was no difference in KLH- or TNP-IgG titers betweenp210 immunized mice and the titers from mice of PBS or cBSA/alum groups(FIG. 8).

Additional examples are described in US provisional Ser. No. 61/261,331and in particular in Appendix A of said provisional which is hereinincorporated in its entirety. In summary in several embodiments,Immunostimulatory T cell, compositions, methods and systems aredescribed for treating and/or preventing atherosclerosis and/or acondition associated thereto in an individual.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the T cell, compositions, systems and methodsof the disclosure, and are not intended to limit the scope of what theinventors regard as their disclosure. Modifications of theabove-described modes for carrying out the disclosure that are obviousto persons of skill in the art are intended to be within the scope ofthe following claims. All patents, patent applications and publicationsmentioned in the specification are indicative of the levels of skill ofthose skilled in the art to which the disclosure pertains. Allreferences cited in this disclosure are incorporated by reference to thesame extent as if each reference had been incorporated by reference inits entirety individually.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference.

Further, the copy of the sequence listing submitted herewith in computerreadable form forms part of the present description and is incorporatedherein by reference in its entireties.

It is to be understood that the disclosures are not limited toparticular compositions or biological systems, which can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting. As used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. The term “plurality”includes two or more referents unless the content clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the disclosure pertains.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of theproducts, methods and system of the present disclosure, exemplaryappropriate materials and methods are described herein as examples.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

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What is claimed is:
 1. A pharmaceutical composition comprising: aquantity of T cells, wherein the T cells consist of ex vivo activatedCD8(+) T cells activated by one or more immunogenic fragments of ApoB100associated with atherosclerosis reduction, wherein the activated CD8(+)T cell is specific for the one or more immunogenic fragments of ApoB100and wherein the one or more immunogenic fragments are selected from SEQID NO: 1 to SEQ ID NO:
 302. 2. The pharmaceutical composition of claim1, wherein the one or more immunogenic fragments is selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 10, (SEQ IDNO: 11, SEQ ID NO: 25, SEQ ID NOs: 30-34, SEQ ID NO: 40, SEQ ID NO: 40,SEQ ID NO: 45, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 94, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, SEQID NO: 107, SEQ ID NO: 111, SEQ ID NO: 129, SEQ ID NO: 143, SEQ ID NO:148, SEQ ID NO: 149, SEQ ID NO: 154, SEQ ID NO: 162, SEQ ID NO: 169, SEQID NO: 177, SEQ ID NO: 199, SEQ ID NO: 210, SEQ ID NO: 222, SEQ ID NO:236, SEQ ID NO: 252, and SEQ ID NO:
 301. 3. The pharmaceuticalcomposition of claim 1, wherein the one or more immunogenic fragments isselected from the group consisting of SEQ ID NO:2, SEQ ID NO: 11, SEQ IDNO: 32, SEQ ID NO: 45, SEQ ID NO: 74, SEQ ID NO: 102, SEQ ID NO: 148,SEQ ID NO: 162, and SEQ ID NO:
 210. 4. The pharmaceutical composition ofclaim 1, wherein the one or more immunogenic fragments comprise apeptide having SEQ ID NO: 143 and a peptide having SEQ ID NO:
 210. 5.The pharmaceutical composition of claim 1, wherein the one or moreimmunogenic fragments comprise a peptide having SEQ ID NO: 11, a peptidehaving SEQ ID NO: 25 and a peptide having SEQ ID NO:
 74. 6. Thepharmaceutical composition of claim 1, wherein the immunogenic fragmentof ApoB100 comprises a peptide having the sequence set forth in SEQ IDNO: 2 or an immunogenic fragment thereof.
 7. The pharmaceuticalcomposition of claim 1, wherein the immunogenic fragment of ApoB100comprises a peptide having the sequence set forth in SEQ ID NO: 45 or animmunogenic fragment thereof.
 8. The pharmaceutical composition of claim1, wherein the immunogenic fragment of ApoB100 comprises a peptidehaving the sequence set forth in SEQ ID NO: 102 or an immunogenicfragment thereof.
 9. The pharmaceutical composition of claim 1, whereinthe immunogenic fragment of ApoB100 comprises a peptide having thesequence set forth in SEQ ID NO: 210 or an immunogenic fragment thereof.10. The pharmaceutical composition of claim 1, wherein the quantity ofCD8+ T cells is about 500,000 to about 2,000,000 activated CD8(+) Tcells.
 11. The pharmaceutical composition of claim 1, wherein thequantity of CD8+ T cells is about 1,500,000.
 12. The pharmaceuticalcomposition of claim 1, wherein the quantity of CD8+ T cells is about1,000,000.